An adjuvant is a substance that enhances the immunogenicity of an antigen. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect, facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system, and may attract immune cells to an antigen depot and stimulate such cells to elicit an immune response.
Recombinant proteins are promising vaccine or immunogenic composition candidates because they can be produced at high yield and purity and manipulated to maximize desirable activities and minimize undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or immunogenic compositions. Such antigens, especially when recombinantly produced, may elicit a stronger response when administered in conjunction with an adjuvant.
Adjuvants have been used for many years to improve the host immune response to antigens of interest in vaccines, especially subunit or component vaccines comprised of recombinant proteins. Intrinsic adjuvants, such as lipopolysaccharides, normally are components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators that are typically non-covalently linked to antigens and are formulated to enhance the host immune response. Aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. Currently, alum is the only adjuvant licensed for human use, although hundreds of experimental adjuvants such as cholera toxin B are being tested. However, adjuvants such as cholera toxin B have deficiencies. For instance, while cholera toxin B is not toxic in the sense of causing cholera, even the most remote chance of minor impurity makes such adjuvants of limited applicability.
Formylmethionyl-peptides are naturally occurring, low molecular weight, biologically active ligands produced by opportunistic enteric bacteria. The most common type of formylmethionyl-peptide is formyl-methionine-leucine-phenylalanine (fMLP). FMLP is a proinflammatory peptide which is able to stimulate many leukocyte functions. It stimulates neutrophil chemotaxis, lysosomal enzyme release, oxygen-free radical production, Ca++ flux, leukotriene release by neutrophils and smooth muscle contraction. fMLP stimulation of neutrophils induces rapid alterations in their expression of adhesion receptors. In addition, fMLP has been shown to induce superoxide production and an increase in intracellular Ca++ levels.
fMLP has been shown to induce chemotaxis in a number of cells, including pulmonary alveolar macrophages, neutrophils, dendritic cells (DC) and monocytes. In fact, the chemotactic or chemoattractant activity of fMLP is sufficiently well established that fMLP is often used as a positive control in chemotactic assays.
Over a decade ago Kashkin et al. (lmunologiya 6: 37-40 (1987)) reported on the immunomodulating activity of fMLP when immobilized with an antigen within a liposome. In effect, Kashkin demonstrated that when fMLP was coimmobilized with bovine serum albumin (BSA) on the surface of liposomes and administered to mice subcutaneously this combination could generate a humoral immune response comparable to that obtained by immunization with BSA plus Complete Freund's Adjuvant. However, this study noted that fMLP and the antigen must be coimmobilized on the surface of liposomes in order for an immunostimilatory or adjuvant activity to be manifested. Significantly, Kashkin et al. reported that administration of fMLP had no immunomodulating effect unless it was formulated together with the antigen on liposomes. FMLP had no effect even when added to suspensions of antigen-loaded liposomes prior to injection. The requirement that the adjuvant and antigen be formulated together on the surface of a liposome makes this approach complicated and of limited applicability.
It would be desirable to enhance the immunogenicity of antigens, by methods other than the use of a conventional adjuvant, especially in monovalent preparations; and, in multivalent preparations, to have the ability to employ such a means for enhanced immunogenicity with an adjuvant, so as to obtain an even greater immunological response. There exists a need for safe and effective adjuvants that can enhance the action of vaccines, especially component vaccines comprised of recombinant proteins and that are easy to prepare and use.